爪蟾卵母：显微注射方法优化，滤泡细胞层的去除，并在电生理实验快速变化的解决方案
Summary
Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.
Abstract
非洲爪蟾卵母细胞作为异源表达系统用于蛋白质，首先由Gurdon 等人所述。 1并已发现以来被广泛使用（参考文献2 – 3，和其中的参考文献）。这使得外国通道表达卵母细胞有吸引力的一个特点是内生的离子通道4的丰度差。这种表达系统已被证明对于许多蛋白质，其中配体门控离子通道的特征是有用的。
GABA A受体在爪蟾卵母细胞和其功能特性的表达被这里描述，包括卵母细胞，用的cRNA，去除滤泡细胞层，并在电生理实验快溶液变更显微注射的隔离。的过程被在这个实验室5,6-优化，从常规使用7-9的那些偏离。传统上，裸卵准备用在RT延长胶原酶处理卵巢裂片的，并且这些裸露的卵母细胞显微注射用的mRNA。采用优化方法，多元膜蛋白已表达并研究了该系统中，如重组GABA A受体10-12，人重组氯通道13，锥虫钾通道14，和一个肌醇转运15,16。
这里详细描述的方法可以应用于在爪蟾卵母选择的任何蛋白质的表达，并迅速解变化可以用来研究其它配体门控离子通道。
Introduction
爪蟾卵母被广泛用作表达系统（参考文献2 – 3，和其中的参考文献）。它们能够正确地组装和整合功能活性多亚基蛋白到其质膜。使用该系统，有可能在功能调查膜蛋白单独使用或与其它蛋白质组合，为了研究突变，嵌合的，或连接在一起的蛋白质的性质，以及筛选潜在药物。
使用的卵母细胞比其它异源表达系统的优点包括巨细胞的简单处理，表达外源遗传信息，卵母细胞的环境的通过浴灌注装置的简单的控制的细胞的比例高，和膜电位的控制。
该表达系统的缺点是在许多实验室17-20中观察到的季节变化。这样做的原因变异目前尚不清楚。此外，卵母细胞的质量经常观察到强烈变化。传统方法7-9包括卵巢瓣的隔离，卵巢瓣的暴露于胶原酶一些小时，裸露的卵母细胞的选择，以及卵母细胞注射。在这里，一些替代方案，快速程序报道，使我们能够用这个表达系统工作超过30年，在卵母细胞质量无季节变化和变化不大。
此处描述了用于卵母细胞的分离，微注射用的cRNA，并除去滤泡细胞层的改性，改进的方法，可用于选择的任何蛋白在非洲爪蟾卵母细胞中的表达。周围的卵母细胞的培养基中快速解的变化非常简单的方法可以应用于任何配体门控离子通道和载流子的的研究。
Protocol
动物实验已通过了伯尔尼州Kantonstierarzt的地方委员会，KantonalerVeterinärdienst伯尔尼（BE85 / 15）。 1. 爪蟾卵母的制备保持在12小时/ 12小时光照/黑暗中水的循环是严格保持在20℃，蛙（ 爪蟾 ）。 从雌蛙9取出卵巢的叶。 注意：凸起的除去是用于再生的刺激，并经常在卵母细胞的质量与手术改善。卵母细胞被卵黄层，卵泡细胞的层，并且含…
Representative Results
爪蟾卵母细胞进行机械挑出使用铂环（ 图1）。的卵母细胞显微注射用的mRNA编码GABA A受体亚单位α4，β2，δ，0.5：0.5：2.5飞摩尔/卵母细胞（ 图2）。后4天，滤泡细胞层除去（ 图3）。卵母细胞电压钳制在-80毫伏和暴露于1μM的3α，21-二羟基5α孕-20-酮（THDOC），一种有效的正变构调节剂的存在增加γ氨基丁?…
Discussion
在这篇文章中描述的方法，从那些传统使用的7-9偏离。它是标准的对卵巢的裂片暴露于1至2小时胶原酶处理8;隔离损坏，裸卵;与使用商业注射装置的mRNA注入它们。此经典程序具有以下缺点：1）卵母细胞很可能是由长曝光于高浓度的胶原酶被损坏。 2）不稳定裸露的卵母细胞必须被存储，直到实验。 3）裸卵更有可能比注射过程中的卵泡挨。商业喷射设备使用大直径（约20微米）注?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the Swiss National Science Foundation grant 315230_156929/1. M.C.M. is a recipient of a fellowship (Beca Chile Postdoctorado from CONICYT, Ministerio de Educacion, Chile).
Materials
| NaCl | Sigma | 71380 | |
| KCl | Sigma | P-9541 | |
| NaHCO3 | Sigma | S6014 | |
| MgSO4 | Sigma | M-1880 | |
| CaCl2 | Sigma | 223560 | |
| Ca(NO3)2 | Sigma | C1396 | |
| HEPES | Sigma | H3375 | |
| Penicilin/streptomycin | Gibco | 15140-148 | 100 μg penicillin/ml and 100 μg streptomycin/ml |
| Platinum wire loop | home-made | ||
| Micropipette puller | Zeitz-Instruments GmBH | DMZ | |
| Hamilton syringe | Hamilton | 80300 | 10 μl, Type 701N |
| Thick walled polytetrafluoroethylene tubing | Labmarket GmBH | 1.0 mm OD | |
| Paraffin oil | Sigma | 18512 | |
| Nylon net, gauge 0.8 mm | ZBF Züricher Beuteltuchfabrik AG | ||
| Borosilicate glass tube | Corning | 99445-12 | PYREX |
| Collagenase NB Standard Grade | SERVA | 17454 | |
| Trypsin inhibitor type I-S | Sigma | T-9003 | |
| EGTA | Sigma | E3389 | |
| Glass capillary | Jencons (Scientific ) LTD. | H15/10 | 1.35 ID mm (for perfusion), alternative company: Harvard Apparatus Limited |
| Borosilicate glass capillary | Harvard Apparatus Limited | 30-0019 | 1.0 OD X 0.58 ID X 100 Length mm (for microinjection) |
| Borosilicate glass capillary | Harvard Apparatus Limited | 30-0044 | 1.2 OD X 0.69 ID X 100 Length mm (for two-electrode voltage clamp) |
| γ-Aminobutyric acid (GABA) | Sigma | A2129 | |
| 3α,21-Dihydroxy-5α-pregnan-20-one (THDOC) | Sigma | P2016 | |
| grill motor | Faulhaber | DC micromotor Type 2230 with gear Type 22/2 | |
| micrometer screw | Kiener-Wittlin | 10400 | TESA, AR 02.11201 |
| Sterile plastic transfer pipettes | Saint-Amand Mfg. | 222-20S |
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Cite This Article
Maldifassi, M. C., Wongsamitkul, N., Baur, R., Sigel, E. Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments. J. Vis. Exp. (118), e55034, doi:10.3791/55034 (2016).